The field of the invention is promoting cell death.
Apoptosis plays a central role in the development and homeostasis of all multi-cellular organisms1-4. Abnormal inhibition of apoptosis is a hallmark of cancer and autoimmune diseases, whereas excessive activation of cell death is implicated in neuro-degenerative disorders such as Alzheimer""s disease5,6. In fact, one mode of action of chemotherapeutic drugs is via the activation of apoptosis; understanding how the cell death program is engaged following an insult, and hence why it fails to be engaged in certain settings, offers a novel approach to overcoming the clinical problem of drug resistance; see, e.g. Makin et al., Cell Tissue Res 2000 July;301(1):143-52 (xe2x80x9cApoptosis and cancer chemotherapyxe2x80x9d).
The mechanism of apoptosis is conserved across species and executed with a cascade of sequential activation of initiator and effector caspases7,8. Caspases, a family of cysteine proteases with aspartate substrate specificity, are produced in cells as catalytically inactive zymogens7. Once activated, the effector caspases are responsible for proteolytic cleavage of a broad spectrum of cellular targets that ultimately lead to cell death.
The Inhibitor of Apoptosis (IAP) family of proteins suppress apoptosis by preventing the activation of procaspases and inhibiting the enzymatic activity of mature caspases9,10. Several distinct mammalian IAPs including XIAP, c-IAP1, c-IAP2, and survivin, have been identified, and they all exhibit anti-apoptotic activity in cell culture9,10. In Drosophila, the anti-apoptotic activity of IAPs is removed by Reaper, Grim, and Hid, all of which appear to act upstream of IAPs and physically interact with IAPs to relieve their inhibitory effect on caspase activation11,12. IAPs are known to be overexpressed in human cancers26-33.
One major caspase activation cascade is triggered by the release of cytochrome c from the intermembrane space of mitochondria13-19 Concurrent with cytochrome c release, another regulator of apoptosis, Smac20 (Second mitochondria-derived activator of caspases) or DIABLO21, is also released from the mitochondria into the cytosol. Smac eliminates the inhibitory effect of multiple IAPs and interacts with all IAPs that have been examined, including XIAP, c-IAP1, c-IAP2, and survivin20,21.
Smac is synthesized as a precursor molecule of 239 amino acids; the N-terminal 55 residues serve as the mitochondria targeting sequence that is removed after import20. The mature form of Smac contains 184 amino acids and behaves as an oligomer in solution20. We recently found that the 2.2 xc3x85 resolution crystal structure of the mature form of Smac reveals an arch-shaped homo-dimer with rich surface features (the atomic coordinates are being deposited with the Protein Data Bank with the accession number 1FEW). The homo-dimeric interface is dominated by hydrophobic residues through van der Waals interactions. Mutations of key residues at the interface disrupted dimer formation and significantly weakened the ability of Smac to induce the activation of procaspase-3 and to promote the enzymatic activity of mature caspase-3. In addition, similar to the Drosophila proteins Reaper, Grim, and Hid, the N-terminal amino acids of Smac/DIABLO were indispensable for its function; in fact, mutation of the very first amino acid rendered the resulting protein completely inactive. The sequence homology among Reaper, Grim, and Hid is restricted to their N-terminal 14 amino acids; deletion of these residues led to loss of interaction with IAPs9 and a fusion protein comprising the N-terminal 37-residue peptide of Hid induced apoptosis in insect cells11. Here we further disclose small peptides, and peptide mimetics that are sufficient to bind IAP, promote activation of procaspase-3 and/or promote apoptosis.
The invention provides methods and compositions for enhancing apoptosis of pathogenic cells. The general method comprises the of contacting the cells with an effective amount of an AV peptoid, wherein the AV peptoid is a peptide comprising AX1, wherein X1 is V, I or L, or a peptide mimetic thereof, which interacts with an Inhibitor of Apoptosis protein (IAP) as measured by IAP binding, procaspase-3 activation or promotion of apoptosis, wherein apoptosis of the pathogenic cells is enhanced.
In some embodiments, the cells are in situ in an individual and the contacting step is effected by administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of the AV peptoid, wherein the individual may be subject to concurrent or antecedent radiation or chemotherapy for treatment of a neoproliferative pathology. In other embodiments, the pathogenic cells are of a tumor selected from the group consisting of breast cancer, prostate cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer, ovarian cancer, renal cancer, hepatoma, melanoma, lymphoma, and sarcoma. In yet other embodiments, the AV peptoid is a peptide comprising AX1X2, wherein X1 is V, I or L and X2 is P or A; particularly, comprising AX1X2, wherein X1 is V and X2 is P.
The subject compositions encompass pharmaceutical compositions comprising a therapeutically effective amount of an AV peptoid in dosage form and a pharnmaceutically acceptable carrier, wherein the AV peptoid is a peptide comprising AX1, wherein X1 is V, I or L, or a peptide mimetic thereof, which inhibits the activity of an Inhibitor of Apoptosis protein (IAP) as measured by IAP binding, procaspase-3 activation or promotion of apoptosis.
In some embodiments, such compositions further comprise an additional therapeutic agent, such as an anti-neoproliferative chemotherapeutic agent, other than the AV peptoid. In other embodiments of such compositions, the AV peptoid is a peptide comprising AX1X2, wherein X1 is V, I or L and X2 is P or A; particularly, comprising AX1X2, wherein X1 is V and X2 is P.
The invention also provides assays for identifying agents which modulates the interaction of an AV peptoid with an IAP, active compounds identified in such screens and their use in the foregoing compositions and therapeutic methods. The general assay comprises the steps of incubating a mixture comprising a subject AV peptoid, a second baculoviral IAP repeat domain (BIR2) of XIAP, and a candidate agent; under conditions whereby, but for the presence of said agent, the peptoid specifically interacts with the BIR2 at a reference affinity; detecting a specific interaction of the peptoid with the BIR2 to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that the agent modulates the interaction of the peptoid to the BIR2 of the XIAP.
In some embodiments of the screen, the detecting step comprises measuring in vitro binding of the peptoid to the BIR2 by pull-down assay, fluorescent polarization assay or solid-phase binding assay. In other embodiments, the mixture further comprises procaspase-3 and a caspase-3 substrate and the detecting step comprises measuring the interaction inferentially by detecting a reaction product of the caspase-3 substrate and caspase-3 generated by activation of the procaspase-3. In yet other embodiments, the incubating step comprises incubating a cell comprising the mixture and the detecting step comprises measuring the interaction inferentially by detecting apoptosis of the cell, particularly wherein the cell is in situ in an animal host.
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean one or more, the term xe2x80x9corxe2x80x9d means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.
An AV peptoid is a peptide comprising AX1, wherein X1 is V, I or L, or a peptide mimetic thereof, which interacts with an Inhibitor of Apoptosis protein (IAP) as measured by IAP binding, procaspase-3 activation or promotion of apoptosis as described in the exemplified activity assays below. In a more particular embodiment, the peptide comprises AX1X2, wherein X1 is V, I or L preferably V and X2 is P or A, preferably P. The subject AV peptoids are fewer than 20 residues (monomers), preferably fewer than 10, more preferably fewer than 5 and preferably 2 or 3 in length, with a molecular weight of less than about m1,000, preferably less than about 500.
AV peptoids include peptide mimetics of the subject peptides. A peptide mimetic is a non-naturally occurring analog of a peptide which, because of protective groups at one or both ends of the mimetic, or replacement of one or more peptide bonds with non-peptide bonds, is less susceptible to proteolytic cleavage than the peptide itself. For instance, one or more peptide bonds can be replaced with an alternative type of covalent bond (e.g., a carbonxe2x80x94carbon bond or an acyl bond). Peptide mimetics can also incorporate amino-terminal or carboxyl terminal blocking groups such as t-butyloxycarbonyl, acetyl, alkyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl, thereby rendering the mimetic less susceptible to proteolysis. Non-peptide bonds and carboxyl- or amino-terminal blocking groups can be used singly or in combination to render the mimetic less susceptible to proteolysis than the corresponding peptide. Additionally, substitution of D-amino acids for the normal L-stereoisomer can be effected, e.g. to increase the half-life of the molecule. Accordingly, the peptide mimetics include peptides having one or more of the following modifications:
peptides wherein one or more of the peptidyl [xe2x80x94C(O)NRxe2x80x94] linkages (bonds) have been replaced by a non-peptidyl linkage such as a xe2x80x94CH2-carbamate linkage [xe2x80x94CH2xe2x80x94OC(O)NRxe2x80x94]; a phosphonate linkage; a xe2x80x94CH2-sulfonamide [xe2x80x94CH2xe2x80x94S(O)2NRxe2x80x94] linkage; a urea [xe2x80x94NHC(O)NHxe2x80x94] linkage; a xe2x80x94CH2-secondary amine linkage; or an alkylated peptidyl linkage [xe2x80x94C(O)NR6xe2x80x94 where R6 is lower alkyl];
peptides wherein the N-terminus is derivatized to a xe2x80x94NRR1 group; to a xe2x80x94NRC(O)R group; to a xe2x80x94NRC(O)OR group; to a xe2x80x94NRS(O)2R group; to a xe2x80x94NHC(O)NHR group, where R and R1 are hydrogen or lower alkyl with the proviso that R and R1 are not both hydrogen; to a succinimide group; to a benzyloxycarbonyl-NHxe2x80x94(CBZxe2x80x94CHxe2x80x94) group; or to a benzyloxycarbonyl-NExe2x80x94 group having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chloro, and bromo; or
peptides wherein the C terminus is derivatized to xe2x80x94C(O)R2 where R2 is selected from the group consisting of lower alkoxy, and xe2x80x94NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl.
Preferred mimetics have from zero to all of the xe2x80x94C(O)NHxe2x80x94 linkages of the peptide replaced by a linkage selected from the group consisting of a xe2x80x94CR2OC(O)NRxe2x80x94 linkage; a phosphonate linkage; a xe2x80x94CH2S(O)2NRxe2x80x94 linkage; a xe2x80x94CH2NRxe2x80x94 linkage; and a xe2x80x94C(O)NR6xe2x80x94 linkage, and a xe2x80x94NHC(O)NHxe2x80x94 linkage where R is hydrogen or lower alkyl and R6 is lower alkyl,
and wherein the N-terminus of the mimetic is selected from the group consisting of a xe2x80x94NRR1 group; a xe2x80x94NRC(O)R group; a xe2x80x94NRC(O)OR group; a xe2x80x94NRS(O)2R group; a xe2x80x94NHC(O)NHR group; a succinimide group; a benzyloxycarbonyl-NHxe2x80x94 group; and a benzyloxycarbonyl-NHxe2x80x94 group having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chloro, and bromo, where R and R1 are independently selected from the group consisting of hydrogen and lower alkyl,
and still further wherein the C-terminus of the mimetic has the formula xe2x80x94C(O)R2 where R2 is selected from the group consisting of hydroxy, lower alkoxy, and xe2x80x94NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl and where the nitrogen atom of the xe2x80x94NR3R4 group can optionally be the amine group of the N-terminus of the peptide so as to form a cyclic peptide,
and physiologically acceptable salts thereof.
An important aspect of the invention is drawn to peptoids comprising N-substituted glycine analogs which resemble naturally-occurring amino acids (i.e., Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr) and comprising the general formula I: XnNRCH2COOXc, wherein the radicals Xn and Xc are either chains of conventional amino acids, chains of one or more N-substituted glycine analogs, or chains in which conventional amino acids and N-substituted glycine analogs are interspersed.
Preferred N-substituted glycine analogs are those in which R is ethyl, prop-1-yl, prop-2-yl, 1-methylprop-1-yl, 2-methylprop-1-yl, benzyl, 4-hydroxybenzyl, 2-hydroxyethyl, mercaptoethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 2-methylthioeth-1-yl, carboxymethyl, 2-carboxyethyl, carbamylmethyl, 2-carbamylethyl, 3-guanidinoprop-1-yl, imidazolylmethyl, or indol-3-yl-ethyl, particularly where R is 2-methylpropyl, benzyl, 2-hydroxyethyl, 2-aminoethyl, or carboxymethyl. The resemblance between amino acid and substitute need not be exact. For example, one may replace lysine with compounds of formula I in which R is aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, or 6-aminohexyl. Serine may be replaced with hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and the like. In general, a conventional amino acid may be replaced with an N-substituted glycine analog having a sidechain of similar character, e.g, hydrophobic, hydrophilic, polar, nonpolar, aromatic, etc.
Monomer refers to a molecule which may be linked to other monomers to form a peptoid. Monomers include amino acid substitutes, which may include N- and/or C-terminal modifications to facilitate linking, for example, leaving or activating groups.
N-substituted glycine analog refers to compounds of the formula RNHxe2x80x94CH2xe2x80x94COOH, where R is as defined above. The salts and esters of these compounds, as well as compounds of the formula bearing standard protecting groups (e.g., Fmoc, t-Boc, and the like) are also considered within the definition of xe2x80x9cmonomerxe2x80x9d and xe2x80x9cN-substituted glycine analogxe2x80x9d unless otherwise specified.
A peptoid of the invention corresponds to a natural peptide if it elicits a biological activity related to the biological activity of the natural protein. The elicited activity may be the same as, greater than or less than that of the natural protein, i.e., provide enhanced and/or blocking effects. In general, such a peptoid will have an essentially corresponding monomer sequence, where a natural amino acid is replaced by an N-substituted glycine derivative, if the N-substituted glycine derivative resembles the original amino acid in hydrophilicity, hydrophobicity, polarity, etc. Thus, the following pairs of peptoids would be considered
IIa: Ala-Ile-Pro-Gly-Phe-Ser-Pro-Phe (SEQ ID NO: 1)
IIb: Ala-Ile-Pro-Gly-Phe*-Ser*-Pro-Phe* (SEQ ID NO: 2)
IIIa: Ala-Leu-Phe-Met-Thr (SEQ ID NO: 3)
IIIb: Ala-Leu-Phe*-Met-Ser* (SEQ ID NO: 4)
In these examples, xe2x80x9cVal*xe2x80x9d refers to N-(prop-2-yl)glycine, xe2x80x9cPhe*xe2x80x9d refers to N-benzylglycine, xe2x80x9cSer*xe2x80x9d refers to N-(2-hydroxyethyl)glycine, xe2x80x9cLeu*xe2x80x9d refers to N-(2-methylprop-1-yl)glycine, and xe2x80x9cIle*xe2x80x9d refers to N-(1-methylprop-1-yl)glycine.
The correspondence need not be exact: for example, N-(2-hydroxyethyl)glycine may substitute for Ser, Thr, Cys, and Met;. N-(2-methylprop-1-yl)glycine may substitute for Val, Leu, and Ile. Note in IIIa and IIIb above that Ser* is used to substitute for Thr and Ser, despite the structural differences: the sidechain in Ser* is one methylene group longer than that of Ser, and differs from Thr in the site of hydroxy-substitution. In general, one may use an N-hydroxyalkyl-substituted glycine to substitute for any polar amino acid, an N-benzyl- or N-aralkyl-substituted glycine to replace any aromatic amino acid (e.g., Phe, Trp, etc.), an N-alkyl-substituted glycine such as N-butylglycine to replace any nonpolar amino acid (e.g., Leu, Val, Ile, etc.), and an N-(aminoalkyl)glycine derivative to replace any basic polar amino acid (e.g., Lys and Arg).
The peptoids of the invention can be produced using amino acids as the monomer units or amino acid substitutes. Examples of different modifications in amino acids which can be carried out in order to obtain the amino acid substitutes used in the invention are put forth below in Table 1.
Items II, III, IV and IX are taken from Spatola, A., xe2x80x9cPeptide Backbone Modifications: . . . xe2x80x9d in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins (1983) 7:267, B. Weinstein ed. The + refers to the extent to which replacement is characterized by the given property: +=minimal, ++=partial, +++=substantial.
As can be seen from the table, modifications can significantly alter the properties of the molecules, particularly with respect to enzymatic hydrolysis. From a synthetic-standpoint, chiral starting materials can be problematic. Even if they are easily synthesized, the fidelity of the subsequent coupling reactions needs to be addressed. Each substitute amino acid structure is discussed briefly below and can be compared to the amino acid structure in a peptide.
I. Peptide. The individual monomer units or substitute peptides such as those described below can be combined together in any manner. However, it is most preferable to combine the monomer units using methodology such as disclosed in WO89/10931 in order to obtain large libraries of different peptoids, which libraries can then be screened to find one or more peptoids which has a particular characteristic such as a high affinity for a particular receptor site. Although the substitute amino acids put forth below are examples of preferred substitute amino acids which can be used in connection with producing peptoids of the invention, it should be noted that any monomer unit can be used which would allow for sequence specific synthesis of pools of diverse molecules. Any such monomer unit would be suitable for use in connection with the present invention in that such units would make it possible to search and screen for particular conformational shapes which have affinity for particular receptor sites. The use of nonpeptide polymers is believed to have particular advantages over conventional peptides in that such peptoids would occupy different conformational configurations in space and should provide resistance to the action of proteases, which feature would be particularly important to designing conjugates wherein the peptoid portion would have a desirably long half-life. Further, substitute amino acids could be designed so as to provide for molecules which are generally easier to synthesize than conventional peptides might be.
II. N-alkylated glycines. The main advantages of this system are the ease of synthesis of the properly protected achiral monomers and the vast literature of peptides concerning the synthesis and characterization of the closely related peptoid polymers. The main disadvantage is the decrease in properties dependent on the availability of amide protons for hydrogen bonding, such as solubility in aqueous systems, conformational rigidity, secondary structure, etc. It is pointed out that N-alkylated glycines are a preferred class of N-substituted glycines which can be used in connection with the present invention. Thus other chemically compatible groups other than R=alkyl may be used. Further, the substitutions may be made on the nineteen other natural amino acids.
III. xcex1-Esters. Polyesters are one of the closest relatives to the normal peptide bonds. The advantage is the close similarity, however, this can also be a drawback since proteolytic enzymes are known to recognize esters or even prefer esters as their substrates. xcex1.-Polyesters are prepared from chiral .alpha.-hydroxy acids in which there has been considerable synthetic interest (Chan, P. C., et al., Tetrahedron Lett (1990) 31:1985). In a stepwise fashion, polymers can be assembled much as polyamides are prepared.
IV. Thioamides. The thioamide is also rather similar to the normal peptide. According to Clausen, K., et al., J Chem Soc Perkin Trans (1984) 1:785, until 1984 there had been only limited reports of the thioamide replacement for a peptide bond which they attribute to the difficulty in synthesis. He describes the synthesis and use of a protected thioamide precursor using Lawessons""s reagent. Also, a recent report (Tetrahedron Lett (1990) 31:23) describes the conversion of a peptide bond to a thioamide using the same reagent.
V. N-hydroxy amino acids. The advantages are the decreased sensitivity to enzyme hydrolysis and H-bonding ability due to the added hydroxyl group. Kolasa et al. has described the synthesis of N-hydroxypeptides (Kolasa, T., et al, Tetrahedron (1977) 33:3285).
VI. xcex2-Ester. This is an example of a homologue of the .alpha.-ester. Presumably the different spacing will confer some special properties such as increased resistance to enzyme hydrolysis or novel conformational flexibility. The appropriate starting materials are readily synthesized (Elliott, J., et al., Tetrahedron Lett (1985) 26:2535, and Tetrahedron Lett (1974) 15:1333.
VII. and VIII. Sulfonamides. The two sulfonamides differ by the positioning of the R group. According to Frankel and Moses (Frankel, M., et al., Tetrahedron (1960) 9:289), the peptide analog, i.e., the 1,4 substituted polymer is not stable under their condensation conditions. Compounds of the type VII are readily obtained from chiral xcex2-amino alcohols (Kokotos, G., Synthesis (1990) 299) while those of the type VIII are achiral and easily synthesized.
IX. Ureas. Ureas are also conveniently synthesized from carboxylic acids and amines using the reagent diphenylphosphoryl azide, DPPA (Shiori, T., et al., J Am Chem Soc (1972) 94:6203, and Bartlett, P., et al., Synthesis (1989) 542). Previously prepared peptoids with a single urea replacement had properties similar to the starting peptide (see reference 1, p. 231). Additionally, since there is still an amide proton available for H-bonding, the solubility properties may be better than for N-alkylated glycines.
X. Urethanes. The structure of a urethane is slightly different than that of a urea and would presumably have altered properties. Aqueous solubility may be somewhat reduced since the amide proton is removed. The polymers may be prepared via simple chemistry.
There are numerous other polymer systems which could be employed for the purpose of searching conformational space. Most notable are the phosphorous derived polymers with phosphonamides as one example (Yamauchi, K., et al., Bull Chem Soc Japan (1972) 45:2528). Polyamines (Tetrahedron Lett (1990) 31:23, and Kaltenbronn, J. S., et al., in Proceedings of the Eleventh American Peptide Symposium (1989) 969, J. Rivier, ed.), polyalkanes, polyketones (Almquist, G., et al., J Med Chem (1984) 27:115, polythioethers, polysulfoxides (Spatola, A., et al., Biopolymers (1986) 25:S229) and polyethers may be less suitable for our purposes due to either difficulty in synthesis or predictably poor properties (e.g., polyamines would carry a positive charge at every junction and require double amine protection during synthesis). In summary, several alternatives to N-alkylated glycine polymers of which libraries could be constructed have been described.
The foregoing examples of mimetics are nonlimiting. Peptide mimetic chemistry is a well-established art wherein skilled practitioners can readily generate a wide variety of mimics using conventional chemistry (see, e.g. Liao et al. (1998) J.Med.Chem 41, 4767-4776; Andrade-Gordon et al. (1999) PNAS USA 96, 12257-12262; Boatman et al. (1999) J.Med.Chem. 42, 1367-1375; Kasher et al. (1999) J.Mol.Biol 292,421-429; U.S. Pat. No. 5,981,467; etc.) and these other strategies are applicable here, so long as the resultant mimetics are screened for and demonstrated to provide the requisite IAP inhibitory activity as assayed below.
Synthetic methods for producing the subject peptoids are well-known in the art. Some general means for the production of peptides, analogs or derivatives are outlined in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, A Survey of Recent Developments, Weinstein, B. ed., Marcell Dekker, Inc., publ. New York (1983). A wide variety of well-established techniques are available for synthesizing peptide mimetics, see, e.g. submonomer method of R. Zuckermann et al., J. Am. Chem. Soc. (1992) 0114:10646-7. Synthesis by solid phase techniques of heterocyclic organic compounds in which N-substituted glycine monomer units forms a backbone is described in U.S. Pat. No. 5,958,792, wherein combinatorial libraries of mixtures of such heterocyclic organic compounds can then be assayed for the ability to inhibit IAP as described below. Highly substituted cyclic structures can be synthesized on a solid support by combining the submonomer method with powerful solution phase chemistry. Cyclic compounds containing one, two, three or more fused rings are formed by the submonomer method by first synthesizing a linear backbone followed by subsequent intramolecular or intermolecular cyclization, also as described in U.S. Pat. No. 5,958,792. General preparative protocols for exemplary peptoid classes are as follows:
Preparation of xcex1-Polyesters Using Chiral xcex1-Hydroxy Acids As Building Blocks. The xcex1-polyester structures can be prepared by using chemical synthesis technology known to those skilled in the art. For details of the reaction, see Brewster, P., et al., Nature, (1990) 166:179. An alternative method for producing similar structures is disclosed in Chan, P. C., and Chong, J. M., Tetrahedron Lett. (1990)1985. Further, various publications cited within the Chan et al. publication describe techniques for synthesizing chiral xcex1-hydroxy acids.
Preparation of Polythioamides Using Chiral .alpha.-Amino Acids As Building Blocks. Polythioamide structures can be synthesized using techniques such as those described in Clausen, K., et al., J. Chem. Soc. Perkin Trans. I (1984) 785, and Tetrahedron Lett. (1990) 31:23.
Preparation of Polyhydroxymates Using Chiral .alpha.-Amino Acids As Building Blocks. Polyhydroxymates can be synthesized using techniques as disclosed in Kolasa, T., and Chimiak, A., Tetrahedron (1977) 33:3285. References cited within Kolasa disclose and describe chemical techniques for synthesizing N-hydroxy amino acids which can be used in peptoid synthesis.
Preparation of xcex2-Polyesters Using Chiral Bxcex2-Hydroxy Acids As Building Blocks. xcex2-polyesters can be synthesized using a synthesis protocol as described in Elliott, J. D., et al., Tetrahedron Lett. (1985) 26:2535, and Tetrahedron Lett. (1974) 15:1333.
Preparation of Polysulfonamides Using Chiral xcex2-Amino Sulfonic Acids As Building Blocks. Polysulfonamides can be synthesized using the reaction scheme shown in U.S. Pat. No. 6,075,121. The chiral xcex2-amino acids have been described within Kokotos, G., Synthesis (1990) 299.
Preparation of N-alkylated Polysulfonamides Using Achiral xcex2-Amino Sulfonic Acids As Building Blocks. Similarly, these polysulfonamides can be synthesized using the reaction scheme shown in U.S. Pat. No. 6,075,121.
Preparation of Polyureas Using Achiral xcex2-amino Acids As Building Blocks. Polyureas can be synthesized using techniques such as those described in Shiori, T., et al., J. Am. Chem. Soc. (1972) 94:6302, and Scholtz, J., and Bartlett, P., Synthesis (1989) 542.
Preparation of Polyurethanes Using Achiral xcex2-Amino Alcohols As Building Blocks. Polyurethanes can be synthesized using the reaction scheme shown in U.S. Pat. No. 6,075,121. Individual N-substituted glycine analogs are known in the art, and may be prepared by known methods. See, for example, Sempuku et al., JP 58/150,562 (Chem Abs (1984) 100:68019b); Richard et al., U.S. Pat. No. 4,684,483; and Pulwer et al., EPO 187,130.
Several N-substituted glycine derivatives are available from commercial sources. For example, N-benzylglycine is available from Aldrich Chemical Co. (Milwaukee, Wis.) as the ethyl ester. The ester is hydrolyzed in KOH/MeOH, then protonated in HCl to yield N-benzylglycine. This may then be protected with Fmoc (fluorenylmethoxycarbonyl) by treatment with Fmoc-Cl in aqueous dioxane at high pH (about 10).
Other N-substituted glycine analogs are synthesized by simple chemical procedures. N-isobutylglycine may be prepared by reacting excess 2-methylpropylamine with a haloacetic acid.
N-(2-aminoethyl)glycine may be prepared by reacting excess 1,2-diaminoethane with a haloacetic acid and purifying on Dowex-1(copyright) (OH form), eluting with acetic acid. The unprotected amine is protected with t-butoxycarbonyl (t-Boc) using conventional techniques at pH 11.2, followed by protection of the secondary amine with Fmoc.
N-(2-hydroxyethyl)glycine may be prepared by reacting excess 2-aminoethanol with haloacetic acid and purifying on Dowex-1(copyright) (OH form), eluting with acetic acid. The amine nitrogen is then protected with Fmoc. Next, the acid group is esterified with methanol under acidic conditions. The methyl ester is then treated with isobutylene to form the t-butyl ether. Then, the methyl ester is hydrolyzed using porcine liver esterase in phosphate buffer at pH 8.0, to provide a protected N-substituted glycine analog in a form suitable for peptoid synthesis. As an alternative to the above, the Fmoc-hydroxyethylglycine is treated with t-butyldiphenylsilylchloride in DMF and imidazole to give a silyl-protected alcohol.
N-(carboxymethyl)glycine may be prepared by reacting glycine t-butyl ester with 2-haloacetate in aqueous solution. The product may be protected directly by addition of Fmoc. As an alternative, the N-(carboxymethyl)glycine may be prepared by mixing glycine t-butyl ester, glyoxylic acid and palladium on charcoal under an atmosphere of hydrogen in water at pH 6. The compound is then treated with FMOC in the usual manner.
Once the monomers have been synthesized, they may be coupled with other monomers and/or conventional amino acids to form analogs using standard peptide chemistry. For example, an Fmoc-protected monomer (N-substituted glycine or conventional amino acid) may be immobilized on a suitable resin (e.g., HMP) by reaction with benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or a carbodiimide (for example, dicyclohexylcarbodiimide) under basic conditions (e.g., pH 9) in a suitable solvent. The Fmoc protecting group is removed by treatment with piperidine. Each additional monomer is then attached sequentially using BOP or a carbodiimide, until the entire sequence has been constructed. The completed chain is then detached from the resin and the sidechain deprotected by treating with trifluoroacetic acid (TFA).
Alternatively, one may connect N-substituted glycine analogs to the ends of peptoids produced by other methods, for example, by recombinant expression or isolation from natural sources. Further, N-substituted glycine analogs may be inserted within the sequence of such peptoids by cleaving the peptoid at the desired position, attaching an N-substituted glycine analog, and reattaching the remainder of the molecule or a chemically-synthesized replacement.
The compositions for administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term xe2x80x9cunit dosage formsxe2x80x9d refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules, losenges or the like in the case of solid compositions. In such compositions, the peptoid is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
Suitable excipients or carriers and methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in such publications as Remington""s Pharmaceutical Science, Mack Publishing Co, NJ (1991). In addition, the peptoids may be advantageously used in conjunction with other chemotherapuetic agents such as diethylstilbestrol or DES, 5-fluorouracil, methotrexate, interferon-alpha, aspariginase, tamoxifen, flutamide, etc, and chemotherapeutic agents described in the Merck Manuel, 16th edition 1992, Merck Research Laboratories, Rahway, N.J.; Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9thEd., 1996, McGraw-Hill, esp. Chabner et al., Antineoplastic Agents at pp .1233, etc. or otherwise known in the art. Hence the agents and peptoids may be administered separately, jointly, or combined in a single dosage unit. In a particular embodiment, the combination therapy is effected by a conjugate of the peptoid bound covalently to the anti-neoproliferative chemotherapeutic or other pharmaceutically active agent. Any suitable conjugation chemistry may be used, such as derivatizing the N-terminus of the peptoids and conjugating the drug through an amid linkage.
The amount administered depends on the AV peptoid formulation, route of administration, etc. and is generally empirically determined in routine trials, and variations will necessarily occur depending on the target, the host, and the route of administration, etc. Generally, the quantity of active compound in a unit dose of preparation may be varied or adjusted from about 0.1 mg to 1000 mg, preferably from about 1 mg to 300 mg, more preferably 10 mg to 200 mg, according to the particular application. The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small amounts until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
The following are examples (Examples 1-4) of capsule formulations for the peptoids of Table 2.
Preparation of Solid Solution
Crystalline peptoid (80 g/batch) and the povidone (NF K29/32 at 160 g/batch) are dissolved in methylene chloride (5000 mL). The solution is dried using a suitable solvent spray dryer and the residue reduced to fine particles by grinding. The powder is then passed through a 30 mesh screen and confirmed to be amorphous by x-ray analysis.
The solid solution, silicon dioxide and magnesium stearate are mixed in a suitable mixer for 10 minutes. The mixture is compacted using a suitable roller compactor and milled using a suitable mill fitted with 30 mesh screen. Croscarmellose sodium, Pluronic F68 and silicon dioxide are added to the milled mixture and mixed further for 10 minutes. A premix is made with magnesium stearate and equal portions of the mixture. The premix is added to the remainder of the mixture, mixed for 5 minutes and the mixture encapsulated in hard shell gelatin capsule shells.
AV peptoids can be administered by a variety of methods including, but not limited to, parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. The chemotherapeutic agent and/or radiation therapy can be administered according to therapeutic protocols well known in the art. It will be apparent to those skilled in the art that the administration of the chemotherapeutic agent and/or radiation therapy can be varied depending on the disease being treated and the known effects of the chemotherapeutic agent and/or radiation therapy on that disease. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of administration) can be varied in view of the observed effects of the administered therapeutic agents (i.e., antineoplastic agent or radiation) on the patient, and in view of the observed responses of the disease to the administered therapeutic agents.
The particular choice of peptoid, chemotherapeutic agent and/or radiation depends upon the diagnosis of the attending physicians and their judgement of the condition of the patient and the appropriate treatment protocol. The peptoid, chemotherapeutic agent and/or radiation may be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, in any order, depending upon the nature of the proliferative disease, the condition of the patient, and the actual choice of chemotherapeutic agent and/or radiation to be administered in conjunction (i.e., within a single treatment protocol) with the peptoid. Similarly, the peptoid and the chemotherapeutic agent do not have to be administered in the same pharmaceutical composition, and may, because of different physical and chemical characteristics, be administered by different routes.
In one embodiment of the present invention, the method of the invention includes systemic or local administration of an AV peptoid. Where systemic administration is desired, the peptoid may be administered, for example, by intravenous injection or orally. One embodiment of the invention provides local administration of the peptoid, for example, at the tumor site. With local administration of the peptoid, the preferred mode of administration is by local injection. However, local administration may also be by catheter, or by local deposition, for example by intra- or peritumoral administration of products sold under the trademark Depofoam(copyright), slow release pump/drug delivery service, implantable or topical gel or polymer, depending on the nature and location of the tumor. Administration of the therapeutics of the invention can also be effectd by gene therapy protocol.
The therapeutics of the invention can be administered in a therapeutically effective dosage and amount, in the process of a therapeutically effective protocol for treatment of the patient. The initial and any subsequent dosages administered will depend upon the patient""s age, weight, condition, and the disease, disorder or biological condition being treated. Depending on the therapeutic, the dosage and protocol for administration will vary, and the dosage will also depend on the method of administration selected, for example, local or systemic administration. For a very potent peptoid, microgram (ug) amounts per kilogram of patient may be sufficient, for example, in the range of about 1 ug/kg to about 500 mg/kg of patient weight, and about 100 ug/kg to about 5 mg/kg, and about 1 ug/kg to about 50 ug/kg, and, for example, about 10 ug/kg.
In general, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect, for each therapeutic, each administrative protocol, and administration to specific patients will also be adjusted to within effective and safe ranges depending on the patient condition and responsiveness to initial administrations. However, the ultimate administration protocol will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as peptoid potency, severity of the disease being treated. For example, a dosage regimen of the peptoids can be oral administration of from 10 mg to 2000 mg/day, preferably 10 to 1000 mg/day, more preferably 50 to 600 mg/day, in two to four (preferably two) divided doses, to reduce tumor growth. In a preferred embodiment, in cases where the peptoid is based on a fused-ring cyclic benzocycloheptapyridine, the preferred dosage of the inhibitor is oral administration of from 50 to 600 mg/day, more preferably 50 to 400 mg/day, in two divided doses. Intermittant therapy (e.g., one week out of three weeks or three out of four weeks) may also be used.
In one example of combination therapy in the treatment of pancreatic cancer, the peptoid is selected from Table 4 or 5, administered orally in a range of from 50 to 400 mg/day, in two divided doses, on a continuous dosing regimen; and the antineoplastic agent is gemcitabine administered at a dosage of from 750 to 1350 mg/m2 weekly for three out of four weeks during the course of treatment. In another example of combination therapy in the treatment of lung cancer, the peptoid is selected from Table 4 or 5, administered orally in a range of from 50 to 400 mg/day, in two divided doses, on a continuous dosing regimen; and the antineoplastic agent is paclitaxel administered at a dosage of from 65 to 175 mg/m2 once every three weeks. In another example of combination therapy in the treatment of gliomas, the peptoid is selected from Table 1, administered orally in a range of from 50 to 400 mg/day, in two divided doses; and the antineoplastic agent is temozolomide administered at a dosage of from 100 to 250 mg/m2. In another example of combination therapy, the peptoid is selected from Table 4 or 5, administered orally in a range of from 50 to 400 mg/day, in two divided doses, on a continuous dosing regimen; and the antineoplastic agent is 5-Fluorouracil (5-FU) administered either at a dosage of 500 mg/m2 per week (once a week), or at a dosage of 200-300 mg/m2 per day in the case of continuous infusion of the 5-FU. In the case of 5-FU administration on a weekly injection, 5-FU may be administered in combination with a foliate agonist, e.g., Leucovoran (at a dosage of 20 mg/m2/week).
A preferred embodiment of the invention includes monitoring the effects of the treatment with an AV peptoid for signs of tumor regression, and subsequently adjusting the administration of further doses accordingly. For example, a person with breast carcinoma would be treated locally with an agent such as cyclophosphamide methotrexate 5-FU (CMF) or tamoxifen or local radiation therapy and an AV peptoid. Subsequent mammography, ultrasound, or physical exams, as compared with the same pre-treatment tests, would direct the course and dosage of further treatment.
The attending clinician, in judging whether treatment is effective at the dosage administered, will consider the general well-being of the patient as well as more definite signs such as relief of disease-related symptoms, inhibition of tumor growth, actual shrinkage of the tumor, or inhibition of metastasis. Size of the tumor can be measured by standard methods such as radiological studies, and successive measurements can be used to judge whether or not growth of the tumor has been retarded or even reversed. Relief of disease-related symptoms such as pain, and improvement in overall condition can also be used to help judge effectiveness of treatment. Accordingly, preferred embodiments of the invention include monitoring of the patient after treatment with an AV peptoid for signs of tumor regression. Such monitoring includes but is not limited to physical exam, CT scan, MRI, mammography, chest X-rays, bone scans, ultra-sounds, bronchoscopy, endoscopy, colonscopy, laparoscopy, and tests for tumor markers such as PSA, CEA, and CA125. The appropriateness of any form of monitoring will be determined by the nature of the cancer being treated.